Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin – Eh Klp5 – led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica . In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2–4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2–4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional.